how does h and e stain work

Full Answer

What is H&E stain and how does it work?

As its name suggests, H&E stain makes use of a combination of two dyes, namely hematoxylin and eosin. This combination deferentially stains various tissue elements and make them easy for observation.

What is the principle of H and E stain?

Principle The principle behind H & E stain is the chemical attraction between tissue and dye. Hematoxylin, a basic dye imparts blue-purple contrast on basophilic structures, primarily those containing nucleic acid moeties such as chromtatin, ribosomes and cytoplasmic regions rich in RNA.

What is the function of eosin stain?

Eosin is anionic and acts as an acidic dye. It is negatively charged and can react with positively charged, acidophilic components in the tissue, such as amino groups in proteins in the cytoplasm. These stain pink as a result. H&E is a useful all-purpose stain that is also quick and easy to use, which may explain why it has stood the test of time.

What is a high-quality H&E stain?

In a high-quality H&E stain, there are subtle differences in the shades of color produced by the stains, particularly eosin, and this aids in the detection and interpretation of morphological changes associated with disease.

How does hematoxylin stain work?

Hematoxylin and eosin (H&E) is the most widely used stain in histology and allows localization of nuclei and extracellular proteins. Hematoxylin, not a dye itself, produces the blue Hematin via an oxidation reaction with nuclear histones causing nuclei to show blue.

What does H&E stain bind to?

(Haematoxylin is not strictly a basic dye, but it is used with a 'mordant' that makes this stain act as a basic dye. The mordant (aluminium salts) binds to the tissue, and then haematoxylin binds to the mordant, forming a tissue-mordant-haematoxylin linkage.)

What is the advantage of H and E staining?

Advantages: H&E yields excellent morphology, providing excellent contrast between cellular components. H&E provides better visualization of individual cell necrosis, keratinization, and keratin pearls, which are frequent histologic findings in welldifferentiated squamous cell carcinoma.

Why do proteins stain with eosin?

These stain blue as a result. Eosin is anionic and acts as an acidic dye. It is negatively charged and can react with positively charged, acidophilic components in the tissue, such as amino groups in proteins in the cytoplasm. These stain pink as a result.

What Colour does H&E stain connective tissue?

pinkIt is typically pink, with different shades of pink for different types of connective tissue fibers.

What are the limitations of H and E staining?

A limitation of hematoxylin staining is that it is incompatible with immunofluorescence. It is useful, however, to stain one serial paraffin section from a tissue in which immunofluorescence will be performed.

How long does H&E staining take?

10 min. maximum for H. then check it under microscope if the stain so dark blue you can use glacial acetic acid to remove the excess of H. And 4 min.

Is hematoxylin basic or acidic?

For example, hematoxylin is an acid, but as it is almost always used in conjunction with alum or iron (the mordant) it becomes a basic stain. Amphophilic - It is a term used to indicate that the tissue stains with both the basic and the acidic dyes.

What does hematoxylin stain in the cell?

Hematoxylin has a deep blue-purple color and stains nucleic acids by a complex, incompletely understood reaction. Eosin is pink and stains proteins nonspecifically. In a typical tissue, nuclei are stained blue, whereas the cytoplasm and extracellular matrix have varying degrees of pink staining.

What does eosin stain in the cell?

Eosin stains the cytoplasm and some other structures including extracellular matrix such as collagen in up to five shades of pink. The eosinophilic (substances that are stained by eosin) structures are generally composed of intracellular or extracellular proteins.

Does haematoxylin stain DNA?

Hematoxylin-based counterstain 4.43. These complexes strongly bind to DNA and RNA, which indicates that it has some carcinogenic properties. When you use a full-strength solution of hematoxylin, the avidity for nucleic acids is so strong that even 10 seconds of exposure will stain the nuclei a light blue.

Which stain is used to stain nucleus?

Cell-Permeant Blue-Fluorescent Counterstains The membrane-permeant Hoechst 33342 dye (H1399, H3570, H21492) has been extensively used for staining the nuclei of live cells.

Why is H&E used in histology?

The H&E staining procedure is the principal stain in histology in part because it can be done quickly, is not expensive, and stains tissues in such a way that a considerable amount of microscopic anatomy is revealed, and can be used to diagnose a wide range of histopathologic conditions.

What is the color of H&E?

H&E is the combination of two histological stains: hematoxylin and eosin. The hematoxylin stains cell nuclei a purplish blue , and eosin stains the extracellular matrix and cytoplasm pink, with other structures taking on different shades, hues, and combinations of these colors. Hence a pathologist can easily differentiate between the nuclear and cytoplasmic parts of a cell, and additionally, the overall patterns of coloration from the stain show the general layout and distribution of cells and provides a general overview of a tissue sample's structure. Thus, pattern recognition, both by expert humans themselves and by software that aids those experts (in digital pathology ), provides histologic information.

What is the H&E method?

The H&E staining method involves application of haematoxylin mixed with a metallic salt, or mordant, often followed by a rinse in a weak acid solution to remove excess staining ( differentiation ), followed by bluing in mildly alkaline water. After the application of haematoxylin, the tissue is counterstained with eosin (most commonly eosin Y ).

What is the most widely used stain in medical diagnosis?

H&E is the combination of two histological stains: hematoxylin and eosin.

What color is hematoxylin?

Hematoxylin principally colors the nuclei of cells blue or dark-purple, along with a few other tissues, such as keratohyalin granules and calcified material. Eosin stains the cytoplasm and some other structures including extracellular matrix such as collagen in up to five shades of pink.

What is the most used stain in Mohs surgery?

Alternatively, H&E stain is the most used stain in Mohs surgery in which tissues are typically frozen, cut on a cryostat (a microtome that cuts frozen tissue), fixed in alcohol, and then stained.

What are eosinophilic structures?

The eosinophilic (substances that are stained by eosin) structures are generally composed of intracellular or extracellular proteins. The Lewy bodies and Mallory bodies are examples of eosinophilic structures. Most of the cytoplasm is eosinophilic and is rendered pink. Red blood cells are stained intensely red.

Principle

Hematoxylin is extracted from the Hematoxylon campechianum tree on which, by oxidation, hematein is produced from hematoxylin, the dye used in the hematoxylin and eosin staining technique. The addition of a mordant allows the hematein to bind to the anionic elements of the tissues.

Procedure

The procedure is simplified into several processes: dewaxing, dehydration, hematoxylin, differentiation, bluish, eosin, dehydration, rinsing, coverslip.

Results and interpretation

Nuclei stain blue, while cytoplasm and extracellular matrix have varying degrees of pink staining. Well-fixed cells show considerable intranuclear detail. The nuclei show different patterns of heterochromatin condensation (hematoxylin staining) specific to the cell type and the cancer type that are very important from a diagnostic point of view.

What is the most commonly used staining system?

The most commonly used staining system is called H&E ( Haemotoxylin and Eosin). H&E contains the two dyes haemotoxylin and eosin.

What kinds of histological stains are there?

Most cells are colourless and transparent, and therefore histological sections have to be stained in some way to make the cells visible. The techniques used can either be non-specific, staining most of the cells in much the same way, or specific, selectively staining particular chemical groupings or molecules within cells or tissues. Staining usually works by using a dye, that stains some of the cells components a bright colour, together with a counterstain that stains the rest of the cell a different colour.

What is hematein used for?

It is used to stain acidic (or basophilic) structures a purplish blue. (Haematoxylin is not strictly a basic dye, but it is used with a 'mordant' that makes this stain act as a basic dye.

What are the components of the cytoplasm that react with acidic dyes?

Acidic dyes react with cationic or basic components in cells. Proteins and other components in the cytoplasm are basic, and will bind to acidic dyes. Another way of saying this is that cytoplasmic proteins are acidophilic (acid liking - i.e. bind to acidic dyes).

What reacts with basic dyes?

Basic dyes react with anionic or acidic components in cells. Nucleic acids are acidic, and therefore bind to basic dyes.

Is cytoplasm a basic dye?

Thus the cytoplasm is stained pink in the picture below, by H&E staining. Haematoxylin can be considered as a basic dye (general formula for basic dyes is:dye + Cl - ).

Liver Tissue Processing and Normal Histology

ARIEF A. SURIAWINATA, ... SWAN N. THUNG, in Surgical Pathology of the GI Tract, Liver, Biliary Tract, and Pancreas (Second Edition), 2009

Fungal Diseases

Although visible with H & E stain, hyphal morphology is best demonstrated with silver or other fungal stains. The viable hyphae are basophilic on H & E stain, whereas necrotic hyphae are hyaline or eosinophilic.

Histologic Features of Pituitary Adenomas and Sellar Region Masses

B.K. Kleinschmidt-DeMasters MD, M.B.S. Lopes MD, PhD, in Practical Surgical Neuropathology: A Diagnostic Approach (Second Edition), 2018

Lung and Pleura

Alexander Craig MackinnonJr., Aliya N. Husain, in Differential Diagnosis in Surgical Pathology (Second Edition), 2010

Tendon Tissue Engineering

Brandon Engebretson, ... Vassilios Sikavitsas, in Tendon Regeneration, 2015

Endocrine System1

Margaret A. Miller, in Pathologic Basis of Veterinary Disease (Sixth Edition), 2017

Pathology

W. Dean Wallace MD, William D. Travis MD, in Murray and Nadel's Textbook of Respiratory Medicine (Sixth Edition), 2016

When was H&E stains invented?

The two stains were independently introduced in 1865 and 1875, respectively, by Böhmer and Fischer. In 1876, Wissowzky described their use in combination as a tissue staining method for staining different materials different colors. Despite its simplicity, this stain has stood the test of time. Even now, over a century later, H&E remains ...

What is the name of the stain that produces different colors of tissue?

Haematoxylin and Eosin. For routine examination, haematoxylin and eos in (H&E) is the stain of choice. This stain produces colors different tissue structures, which would otherwise be transparent, so that you can get a detailed view of the tissue. As its name suggests, H&E stain makes use of a combination of two dyes – haematoxylin and eosin.

What color would it be if you viewed an unstained tissue section under a microscope?

If you viewed an unstained tissue section under the microscope, everything would appear a uniform dull grey color. The staining process therefore makes use of various dyes that stain particular cell components within tissues, so that you can distinguish different cell parts from each other.

Why do you need to stain tissue?

This is because unstained tissue lacks contrast: all of the fixed materials have a similar refractive index and a similar color. If you viewed an unstained tissue section under the microscope, everything would appear a uniform dull grey color.

Is haematoxylin a dye?

Haematoxylin alone is not technically a dye, and will not directly stain tissues. It therefore needs to be used in combination with a “mordant” – a compound that helps it link to the tissue. The mordant used is typically a metal cation, such as aluminium. Haematoxylin in complex with aluminium salts is cationic and acts as a basic dye.

Is eosin an acidic dye?

These stain blue as a result. Eosin is ani onic and acts as an acidic dye. It is negatively charged and can react with positively charged, acidophilic components in the tissue, such as amino groups in proteins in the cytoplasm. These stain pink as a result.

Does H&E staining produce color?

The Science Behind H&E. Staining does not produce color randomly; instead, the dyes exploit differences in the chemistry of the tissue to differentially color various components. Ionic bonding is the most important type of bonding that occurs in histologic staining techniques.

Why is H&E stain unchanged?

The stain has been unchanged for many years because it works well wi …. INTRODUCTIONHematoxylin and eosin (H&E) stains have been used for at least a century and are still essential for recognizing various tissue types and the morphologic changes that form the basis of contemporary cancer diagnosis. The stain has been unchanged for many years ...

What is hematoxylin and eosin staining?

Hematoxylin and eosin staining of tissue and cell sections. INTRODUCTIONHematoxylin and eosin (H&E) stains have been used for at least a century and are still essential for recognizing various tissue types and the morphologic changes that form the basis of contemporary cancer diagnosis. The stain has been unchanged for many years ...

Why is hematoxylin blue?

Hematoxylin has a deep blue-purple color and stains nucleic acids by a complex, incompletely understood reaction.

What is the limitation of hematoxylin staining?

A limitation of hematoxylin staining is that it is incompatible with immunofluorescence.

What type of stain is used for nuclei?

Nuclei show varying cell-type- and cancer-type-specific patterns of condensation of heterochromatin (hematoxylin staining) that are diagnostically very important. Nucleoli stain with eosin. If abundant polyribosomes are present, the cytoplasm will have a distinct blue cast.

Is hematoxylin a counterstain?

Hematoxylin, generally without eosin, is useful as a counterstain for many immunohistochemical or hybridization procedures that use colorimetric substrates (such as alkaline phosphatase or peroxidase). This protocol describes H&E staining of tissue and cell sections.

Why use H&E stain?

While both stains are used in histopathology laboratories, H&E stain is more commonly used by pathologists and researchers for investigating underlying cellular and tissue structures. H&E stain’s simplicity, and ability to clearly elucidate the tissue’s basic morphology by staining nuclei and cytoplasm in different colors makes it ideal as ...

What is the purpose of H&E staining?

Routine H&E (Haemotoxylin and Eosin stains) and special staining comes especially handy when examining tissue structure and cell types and/or when looking for the presence of certain microorganisms in a sample. While both stains are used in histopathology laboratories, H&E stain is more commonly used by pathologists and researchers for investigating underlying cellular and tissue structures.

What color is H&E?

and heterochromatin clearly visible in nuclei. H&E staining makes use of two dyes – hematoxylin (a natural basic dye extracted from the logwood tree) and eosin (an acidic dye that gives a bright pink color). This combination works perfectly together since the dyes stain different tissue elements – hematoxylin stains the acidic (basophilic) ...

What are the different types of stains?

There are a lot of special stains available. There are those that can be used for the detection of microorganisms (gram staining, giemsa stain, acid fast blue, acid fast green, etc.), connective tissues and lipids (toluidine blue, elastic stain, gomoris trichrome blue, gomoris trichrome green, Massons trichrome stain, etc.) and carbohydrates (mucicarmine stain, alcian blue, acid-schiff, alcian blue PAS). There are also special stains that can be used to detect minerals (iron stain, von Kossa stain, colloidal iron) and pigments in specimens.

How to stain slides?

In regressive staining, the slides containing the samples to be stained are submerged in the solution for about 5 minutes to saturate all the available binding sites . Excess stain is removed by a “differentiation” step. Here the slides are washed in an acidic solution composed of either dilute HCL or acetic acid in water or alcohol. How fast this acid wash works will vary greatly depending on whether HCL or acetic acid was used, its concentration, and alcohol content (more alcohol slower destaining). The pathologist will then destain until the intensity is adequate, which can sometimes be a subjective matter where personal preference comes into play. The slides are then “blued” by rinsing in tap water until the sections turn blue. While tap water usually provides the alkalinity needed for the bluing process, this varies by location and other alternatives such as “Scott’s water substitute” or ammonia solution can be used. To differentiate the nucleus from the non-nuclear components, the slides are counterstained with 1% eosin for 10 minutes. Finally, the slides are dehydrated in increasing concentrations of alcohol, cleared in xylene and mounted in mounting media for observation.

What is special staining?

By definition, special stains refer to a wide variety of alternate staining techniques and procedures used to provide more in-depth information on a particular sample. Rather than specific antibody binding, special staining techniques are based on simple chemical reactions such as acid-base chemistry and oxidation-reduction reactions.

What is progressive stain?

Progressive stains are those where the intensity progressively increases with staining time. The process has to be interrupted at the right time in order to achieve the desired intensity.

Overview

Hematoxylin and eosin stain (or haematoxylin and eosin stain or hematoxylin-eosin stain; often abbreviated as H&E stain or HE stain) is one of the principal tissue stains used in histology. It is the most widely used stain in medical diagnosis and is often the gold standard. For example, when a pathologist looks at a biopsy of a suspected cancer, the histological section is likely to be stained with H&E.

Uses

Method of application

Results

Mode of action

Further reading

External links